G+c rich binding protein

ABSTRACT

A novel transcription regulator of G+C-rich promoters, the murine G+C-rich Promoter Binding Protein (mGPBP), and its interaction pattern with other transcription factors are disclosed. A human homologue of mGPBP, hGPBP, is described as well.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.10/947,997, filed Sep. 22, 2004, which is a continuation-in-part ofInternational Application No. PCT/US03/07870, filed Mar. 17, 2003, whichclaims priority to U.S. Provisional Application No. 60/366,898, filedMar. 22, 2002. All of the above priority documents are incorporatedherewith by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is generally related to the modulation oftranscription. More specifically, the present invention is related tothe regulation of genes controlled by G+C-rich promoters.

2. Description of Related Art

Promoters that govern the transcription of mammalian genes by RNApolymerase II fall broadly into three types: the classicalTATAAA-box-dependent promoters, the initiator element (Inr)-dependentpromoters, and the G+C-rich promoters which also have been referred toas CpG islands. Transcription initiation at the TATAAA-box-dependentpromoters is dictated by the direct interaction of the TATAAA-box withthe TATA-binding protein TBP as a first and rate-limiting step (reviewedby Buratowski et al., 1989). Likewise, transcription initiation atInr-dependent promoters occurs when the Inr element interacts withsequence-specific Inr-binding proteins (Smale & Baltimore, 1989, Smaleet al., 1990). The mechanism by which transcription initiation siteswithin G+C-rich promoters are recognized by the RNA Polymerase IItranscription machinery is currently unelucidated.

Transcription initiation of a large number of mammalian genes, includingmost housekeeping genes, and many highly regulated genes controllingcell growth and differentiation, is under the control of G+C-richpromoters (Rauth et al., 1989). This class of promoter is not found ineither the drosophila or yeast genomes. Because a common characteristicof this type of promoter is the presence of a non-canonical TATAAA boxand one or more Sp1 binding sites upstream of the major transcriptioninitiation site, several reports have claimed that those sequences arethe key functional elements in G+C-rich promoters (Blake et al., 1990,Innis et al., 1991). However, reports that challenged these claims, insome cases even with respect to the same promoter, also have beenpublished (Means & Farnham, 1990a, 1990b, Ackerman et al., 1993). Thelack of obvious conserved sequence motifs shared among differentG+C-rich promoters, with the exception of the Sp1 binding sites and the“non-canonical” TATAAA boxes, provides further impetus forinvestigations to elucidate how transcription can initiate non-randomlyat these precise genome locations.

SUMMARY OF THE INVENTION

The present invention is related to an isolated DNA encoding a GPBPpolypeptide. The encoded polypeptide may comprise the sequence set forthin SEQ ID NO:2, SEQ ID NO:4 or homologs thereof. The DNA may comprisethe sequence set forth in SEQ ID NO:1 or SEQ ID NO:3. The encodedpolypeptide may specifically bind to a G+C-rich promoter, which may beMSPE. The present invention is also related to an isolated GPBPpolypeptide.

The present invention is also related to a vector comprising a DNAencoding a GPBP polypeptide. The DNA encoding a GPBP polypeptide may beoperative linked to a control group. The vector may be an expressionvector.

The present invention is also related to an antibody that specificallybinds to a GPBP polypeptide. The antibody may be a monoclonal antibodyor polyclonal antibody. The antibody may also be a single chain antibodyor a humanized antibody.

The present invention is also related to a method of detecting alteredexpression of GPBP. A sample to be tested is contacted with an antibodythat specifically binds to a GPBP polypeptide. The binding of theantibody to the sample is measured and compared to a control. Alteredexpression of GPBP is identified by a difference in binding of antibodyto the sample and the control. The present invention is also related toa method of diagnosing cancer by detecting the altered expression ofGPBP.

The present is also related to a method of modulating the stability of aDNA nanostructure comprising adding a GPBP polypeptide to thenanostructure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the sequence of mGPBP and sequence homology. FIG. 1Ashows the complete cDNA sequence corresponding to the 3.0-kb murine GPBPmRNA (SEQ ID NO:1), including the deduced amino acid sequence encoded bythe ORF (SEQ ID NO:2) (GenBank accession number AY382529). FIG. 1B showssequence small regional sequence homology (open boxes) of the ORF (solidbox) on the amino acid sequence level with yeast TFIIFα subunit(TFIIFα), yABF-1, murine SSRP (mHMG-1 like) and helicases.

FIG. 2 shows northern blot analyses to examine the murine GPBP mRNAspecies and tissue distribution. Approximately 3 μg of poly A⁺ RNAderived from the tissues indicated was loaded per lane. The blot shownin panel A was first probed with a mGPBP specific probe (upper panel),stripped, and reprobed with a β-actin probe (bottom panel). In panel B,the blot was probed with a 2.0 kb probe derived from the 5′ terminus ofthe mGPGP cDNA (left panel), stripped, and reprobed with a 350 bp probederived from the 3′ terminus of the mGPBP cDNA corresponding to the 3.5kb mGPBP mRNA (right panel). The 5′ probe hybridized with both mRNAspecies, whereas the 3′ probe hybridized with only the 3.5 kb mRNA.

FIG. 3 shows that mammalian GPBP is approximately 66 kd in size and isubiquitously expressed. Panel A, Lanes 1-5 displayed electrophoreticallyseparated proteins in SDS polyacrylamide gels stained with CoomassieBlue. Bacterial lysate derived a BL21 strain carrying the PET-mGPBPexpression plasmid were analyzed either without (−) or with (+) IPTGinduction. Lane 4 displayed purified recombinant mGPBP. Molecular weightmarkers are shown in lanes 1 and 5. The samples used in lanes 3 and 4were also analyzed by Western blotting using antiserum against mGPBP asprobe in lanes 7 and 8, respectively. Control BL21 cell (without thePET-mGPBP expression plasmid) lysate was analyzed in lane 6. Panel Bshows the Western blot analyses of cell lysates derived from human HeLacells (lane 1), mouse C1-1D cells (lane 2), human JEG-3 cells (lane 3),human VA-2 cells (lane 4), mouse M2-10B4 cells (lane 5), and human 293cells (lane 6) using the anti-mGPBP antiserum as probe.

FIG. 4 shows that the purified recombinant mGPBP can bind specificallyto the mouse ADA gene's G+C-rich promoter in EMSA. The DNA probe 4C′used in panel A is 4 copies of the MSPE C′ that had been end-ligated.Purified rmGPBP binds specifically to the probe and caused a shift inprobe electrophoretic mobility from the free probe location (lane 1) tothe bound probe location (lane 2). This binding can be specificallycompeted out by adding 35-fold (lane 3) and 175-fold (lane 4) excessunlabeled probes, but cannot be competed out by adding similar amountsof unlabeled E2F binding motifs (lanes 5 and 6) or a 200 bp plasmidsequence (lanes 7 and 8). A single copy of this fragment C′ in thecontext of the labeled 236 bp mouse ADA gene promoter can also bind toand be electrophoretically retarded by the purified rmGPBP (Panel B,lanes 1 and 2). This binding can be competed out by excess unlabeledprobe (lanes 3 and 4) or the 4C′ probed used in panel A (lanes 5 and 6).This binding is again not competed out by unlabeled E2F bindingsequences (lanes 7 and 8) or the 200 bp plasmid sequences (lanes 9 and10).

FIG. 5 shows that both human and transfected mGPGP are localized in thenucleus. Human HeLa cells were stained with antibody against mGPBP (N80)(panel A top) or from preimmune serum (panel B top). The location of thenucleus was determined by counterstaining the cells with DAPI (panelsA&B bottom). The transfected HA-tagged mGPBP was localized in panel Cusing the N80 (panel C top) and anti-HA antibody (panel C bottom).

FIG. 6 shows that the mGPBP can complex with multiple transcriptioninitiation complex assembly specific factors. (A) Mouse C1-1D cellnuclear extract proteins were allowed to bind to bead immobilizedGST-mGPBP fusion proteins. The various proteins, including the startingnuclear extract (Input), proteins that bind to bead immobilized GST orGST-mGPBP fusion proteins (Bound to), and proteins that did not bind tobead immobilized GST or GST-mGPBP fusion proteins (Unbound Supernatant)were analyzed by Western blotting. The antibodies used to probe theseblots were raised against TBP, The C-terminal domain of RNA polymeraseII (RNA pol II CTD), Transcription Fraction IIB (TFIIB), TranscriptionFraction IIF RAP30 subunit (TFIIF RAP30), P300/CAAT Binding Protein(CBP), and the negative control antibodies against the nuclear envelopeprotein Nucleoporin p62. The estimated molecular weight of proteins ineach band (in Kd) based on electrophoretic mobility in comparison toprotein size markers were shown on the right by arrows. (B) In vivobinding of mGPBP was determined by immuno-coprecipitation analyses ofcell lysates. Western blot analyses showed C1-1D cell lysate proteinsafter the cells were transfected with expression vectors that expresseither the HA-tag (HA) alone, or the HA-tag fused to mGPBP (HA-mGPBP),without immunoprecipitation (input lysate), or after immunoprecipitationwith antibodies against the HA-tag (IP αHA Ab). The antibodies raisedagainst TBP, RNA pol II CTD, TFIIB, CBP, or HA-tag were used as probesas indicated in each blot. The estimated molecular weight of proteins ineach band (in Kd) based on electrophoretic mobility in comparison toprotein size markers are shown on by arrows. The migration location ofthe HA-mGPBP fusion protein is also shown on the right of the panel withan arrow.

FIG. 7 shows that mGPBP is specifically required for transcriptioninitiated at the mouse ADA gene's G+C-rich promoter. Co-transfection ofa mouse ADA gene promoter controlled luciferase reporter gene constructtogether with increasing amounts of a mGPBP expression vector intoeither mouse C1-1D cells or human 293 cells resulted in a linearincrease in reporter gene activity (Panel A). In vitro transcriptionassays performed by co-incubation of a supercoiled mouse ADA genepromoter controlled G-less cassette reporter gene with human mouse C1-1Dcell nuclear extracts showed that reporter transcript (Transcript)production was unaltered by the addition of increasing amounts ofpre-immune serum derived antibodies (lanes 1-3, Panel B). In contrast,increasing amounts of anti-mGPBP antibodies caused a gradual decrease inreporter transcript production (lanes 4-9, Panel B). Thisimmuno-suppression effect can be reversed by the addition of purifiedrmGPBP (lanes 10 and 11, Panel B). Transcription of the G-less cassettereporter gene under the control of the TATAAA-box dependent adeno-virusmajor late promoter was not affected by the addition of anti-mGPBPantibodies (lanes 12-15, Panel B). The assay results were repeated atleast 3 times and the normalized quantitation of the transcription assayresults (using a phosphorimager), together with standard deviations ofmultiple repetitions are shown in graphic form in panel C.

FIGS. 8A-8B show the complete cDNA sequence of human GPBP (SEQ ID NO:3),including the deduced amino acid sequence encoded by the ORF (SEQ IDNO:4).

FIG. 9 shows that the TATA box contained the Topo IIα gene's G+C-richpromoter binds specifically to mGPBP but is only partially dependent onits presence for promoter function. (A) The human Topo IIα gene promoter(SEQ ID NO:12) shows no obvious sequence homology to the MSPE within theAda gene promoter (SEQ ID NO:13). The sequences displaying an imperfectdyad symmetry flanking the major transcription initiation site (arrow)are underlined. The consensus TATA element in the Topo IIα gene promoteris boxed. (B) Electrophoretic mobility of the ³²P-labeled Topo IIα genepromoter probe (lane 1, arrow) was retarded (bound probe) by thepresence of purified recombinant mGPBP (lane 2). This retardation of theprobe can be reversed by competition with excess unlabeled probe (lanes3 and 4) or linked quadruple copies of the MSPE derived from the Adagene promoter (lanes 7 and 8), but not by excess copies of a 200-bpplasmid sequence that shows no secondary conformational changes undernegative superhelicity (lanes 5 and 6). (C) In vitro transcription ofsupercoiled reporter genes driven by the Topo IIα gene promoter withHeLa nuclear extract was partially suppressed by the presence ofanti-GPBP antibodies but was unaffected by the presence of preimmuneantibodies. The suppressive effect of the anti-GPBP antibodies can befully reversed by the addition of 20 ng of purified recombinant mGPBP tothe reaction mixture. The statistical significance (P) for thedifference of transcription levels in the absence and presence ofanti-mGPBP antibodies (*) was found by a paired Student t test to be<0.000003 (n=5), and P for the difference of transcription levels in thepresence of the anti-mGPBP antibodies with and without additionalrecombinant mGPBP (+) was found by a paired Student t test to be <0.028(n=5).

DETAILED DESCRIPTION

G+C-rich promoters control the transcription initiation of manymammalian genes, including most housekeeping and Central Nervous System(CNS) genes, and many highly regulated genes that control cell growthand differentiation (Melton et al., 1984, McGrogan et al., 1985,Kreidberg & Kelly, 1986, Rauth et al., 1989, Sehgal et al., 1988,Ackerman et al., 1993). Except for the presence of a non-canonicalTATAAA box and one or more Sp1 binding sites, there is no obvioussequence homology that is shared among the many G+C-rich promoters thathave been analyzed. The murine ADA gene's G+C-rich promoter exhibits allthe typical features of this class of promoter. We have previously notedthat the murine ADA gene's MSPE shares no significant sequence homologywith other G+C-rich promoters, including that of the human ADA gene(Ackerman et al., 1993). Our group also demonstrated that neither thenon-canonical TATAAA motif (TAAAAAA) nor the Sp1 binding sites arerequired for basal promoter activity (Ackerman et al. 1993). Morerecently, we have shown that this TAAAAAA sequence is also not requiredfor this promoter to respond to the ADA gene's T-cell specific enhancer(Hsu et al., submitted). It is therefore unclear what signalingmolecules or DNA sequences are required for RNA polymerase IItranscription initiation to occur non-randomly at these precise G+C-richpromoter locations within the mammalian genome.

The search for a DNA binding factor that can recognize a small DNAbinding motif within a G+C-rich promoter and initiate the assembly ofthe transcription initiation complex has been the subject of intensivebut, as yet, unfruitful investigation (Lichtsteiner et al., 1987, Soptaet al., 1989, Means & Farnham, 1990a). Our work on the murine ADA gene'sG+C-rich promoter has established that this promoter has neither afunctional TATAAA-box nor a potential initiator element that is largerthan 4 bp (Ackerman et al., 1993; Hsu et al., submitted). Instead, theminimal self-sufficient promoter activity resides within a 48 bp MSPEwhose sequence displays an imperfect dyad symmetry with theoreticalsecondary structure-forming potential. DNA sequences with thetheoretical potential to form similar structures have also been found inmany other G+C-rich promoters (Ackerman et al., 1993). More recently, wehave shown that this MSPE can adopt a non-B-form DNA secondary structureunder negative-superhelical torsion condition that approximates thatfound in physiological chromatin (Hsu et al., submitted). Since wepreviously had demonstrated that the ADA gene's MSPE contains nuclearprotein binding sites (Ackerman et al., 1993), this DNA element was usedto isolate a cloned nuclear protein that can bind both to the promoterand to key proteins that participate in the assembly of the RNApolymerase II transcription initiation complex. Our success in thisendeavor may owe much to our choice of using this rather large MSPE asthe probe. This MSPE does not depend on other proximal activator motifssuch as Sp1 binding sites to exhibit promoter function (Ackerman et al.,1993). Smaller elements within the MSPE do not suffice asself-sufficient promoters (Ackerman et al., 1993, and Ackerman & Yeung,unpublished data). By using a multimerized MSPE probe, we successfullyidentified a λgt11 phage cDNA expression clone that encodes a G+C-richPromoter Binding Protein (GPBP). Full-length cDNA clones correspondingto the two cross-hybridizing mRNA species, as determined by Northernblot analysis (FIG. 2), were generated using 5′ and 3′ RACE. These 2mRNA species apparently differ only in their 3′ polyadenylation siteutilization, and they both contain an identical 1,479 nt Open ReadingFrame (ORF) 1.15 kb downstream of the transcription initiation site. Theextremely high nucleotide sequence conservation between the 5′ and 3′UTRs of the mouse and human GPBP genes (FIGS. 8A-8B) suggests that theseUTR sequences have important functional roles.

Because G+C-rich promoters are present upstream of many essentialmammalian housekeeping genes, but are not found in either the yeast ordrosophila genomes, we predicted that if GPBP expression is critical totranscription from G+C-rich promoters, it would be ubiquitouslyexpressed in mammalian cells and be absent from both yeast anddrosophila genomes. Northern (FIG. 2) and Western (FIG. 3B) blotanalyses of various mouse tissues and mammalian cell lines confirmed theformer prediction. A sequence homology search of the DNA sequencedatabase (which includes the entire yeast and drosophila genomesequences) revealed no obvious mGPBP orthologs in any of thenon-mammalian genomes, although small domain-specific homologies to anumber of transcription factors were found (FIG. 1B). Thedomain-specific homology to the SSRP1 gene (FIG. 1B) is particularlyintriguing. Since SSRP1 is known to bind DNA in a DNAstructure-dependent manner (Shirakata et al., 1991, Bruhn et al., 1992),it is possible that GPBP may also share that unusual property and bindto MSPE via structural recognition.

Surprisingly, although no obvious mGPBP orthologs were uncovered by thesequence search, the murine ADA gene's G+C-rich promoter does functionwhen it is introduced into yeast cells (data not shown). We note withinterest that yeast ABF-1, which shares some domain-specific homologywith mGPBP (FIG. 1B), can reportedly transactivate the yeast TRP3 genepromoter which possesses a “suboptimal” TATA-box (Martens & Brandl,1994). Because yeast origins of replication do not function as such inmammalian cells and no one has been able to identify a mammalian yABF-1ortholog by low-stringency hybridization screening, yABF-1 's ability totransactivate a yeast gene promoter with a non-canonical TATA-box andits shared sequence homology with certain mGPBP domains raises thepossibility that yABF-1 and mGPBP may share similar cellular functionsbut recognize and bind to highly divergent DNA sequences. It iscertainly formally possible that yABF1, which has no known mammalianorthologs, may also promote transcription at the mouse ADA gene'sG+C-rich promoter in mGPBP-deficient yeast cells.

The predicted size of mGPBP based on the cDNA ORF sequence was confirmedby Western blot analysis of lysates of various mammalian cell linesderived from diverse tissues of origin (FIG. 3). The otherimmuno-cross-reacting proteins seen in all cell lysates (FIG. 3B) may beeither degradation products of mammalian GPBP or other members of a GPBPgene family. We have also cloned the human GPBP ortholog (FIGS. 8A-8B)and found that it contained an ORF that shares 93% homology at thenucleotide level (and 95% identity in the predicted amino acid sequence)with the mouse GPBP ORF. The presence of an extremely well-conservedGPBP gene in the mouse and human genomes and the absence of an orthologgene identifiable by sequence homology in the yeast and drosophilagenomes suggest that the GPBP gene may be one of the very raremammal—(or higher eukaryote—) specific genes. This observation isconsistent with the observation that while mammalian genomes possessnumerous G+C-rich promoters, no such promoters have been reported ineither the yeast or drosophila genome.

Purified recombinant mGPBP was shown to bind specifically to the murineADA gene's MSPE in EMSA in the absence of any other mammalian proteins.In the screening of the cDNA library and in EMSA, a probe consisting of4 copies of the 48 bp MSPE (C′) [which differs from fragment C by onlythe terminal nucleotide at each end] end-ligated into a single 200 bpprobe binds specifically to recombinant mGPBP. The specificity of thebinding reaction was demonstrated in competition EMSAs using specificand non-specific competitor DNA (FIG. 4A). Interestingly, a single copyC or C′ probe, which cannot maintain a potential double stranded 4-wayjunction because of the lack of additional complementary sequencesflanking the potential stem-and-loop structure (Ackerman et al., 1993)did not bind GPBP in similar EMSA experiments (data not shown). However,the 236 bp mouse ADA gene promoter with its single copy of the MSPEeffectively and specifically bound to the recombinant mGPBP (FIG. 4B).The mGPBP binding interaction was specific to the MSPE, since thisbinding can be competed out by excess amounts of the 4C′ fragment, butnot by a similar excess of non-specific DNA sequences. These resultsthus established that purified recombinant mGPBP can specifically anddirectly bind to the murine ADA gene promoter's G+C-rich MSPE in theabsence of any other transcription factors. This observation isconsistent with GPBP potentially being responsible for mediating theinitial assembly of the transcription initiation complex. This GPBP/DNAinteraction cannot be due to junction sequence artifacts, since probes(such as C, C′, and the entire promoter) consisting of MSPE withdifferent flanking sequences all bind effectively and specifically tothe protein. Interestingly, optimal electrophoretic mobility shift ofthe mGPBP-bound MSPE-containing probes occurred in EMSAs performed in1×TBE instead of the standard lower 0.5×TBE salt conditions (FIG. 4, anddata not shown). The higher salt condition should favor the maintenanceof DNA double-strandedness in the MSPE flanking sequences and/or theimperfectly matched potential stem sequence described by Ackerman etal., 1993, and stabilize the unusual non-B form DNA secondary structurethat may be required for mGPBP binding.

In situ binding assays using anti-mGPBP antiserum as a probedemonstrated that endogenous human GPBP is nuclear-localized in HeLacells (FIG. 5A). The nuclear-localization property of the mGPBP wasconfirmed by transfecting HA-tagged mGPBP expression vectors into theHeLa cells and detecting the transduced HA tag in the cell nuclei usinganti-HA antibodies (FIG. 4C). These results are consistent with GPBP'sproposed role as a mediator of transcription initiation complex assemblyat the promoter, a role which would require nuclear-localization.

Western blot analysis of mouse C1-1D cell nuclear extract proteins thateither complex with immobilized mGPBP or are co-immunoprecipitated withHA-tagged mGPBP expressed in intact cells revealed that mGPBP cancomplex specifically with TBP, TFIIB, TFIIF, RNA polymerase II, andP300/CBP both in vitro and in vivo. In contrast, the nucleoporin p62protein, which is present in nuclear extract but does not participate intranscription complex formation, did not complex with mGPBP in the sameassays. Since all these mGPBP-associated transcription initiationfactors are known to participate in the formation of the RNA polymeraseII transcription initiation complex (Buratowski et al., 1989, Chan & LaThangue, 2001), these observations are consistent with the proposed roleof GPBP as a mediator of transcription initiation complex assembly,following its binding to G+C-rich promoters.

In co-transfection assays, mGPBP was found to trans-activate the murineADA gene's G+C-rich promoter (FIG. 7A) in the two different cell lines(C1-1D and 293) used. Because all mammalian cells tested containsignificant levels of endogenous GPBP (FIG. 3B), it is not surprisingthat the transactivating effect of the exogenous recombinant mGPBP inthe transfected cells is only in the 2.5-fold range. More significantly,in the presence of high copy numbers of the co-transfected reporter genein both C1-1D and 293 cells, the trans-activating effect of the mGPBPexpression vector remained linear over the >8-fold range of levels ofGPBP expression vector used (FIG. 7A). Thus, in the presence of anexcess G+C-rich promoter sequences in the cell, GPBP appeared to be ratelimiting for reporter gene transcription. Moreover, GPBP activity wasshown to be essential for transcription directed by the mouse ADA gene'sG+C-rich promoter. In in vitro transcription assays, sequestering ofGPBP in human HeLa cell nuclear extract by immunoabsorption caused aproportional and ultimately complete suppression of transcription fromthe ADA gene's G+C-rich promoter (with the level of transcriptiondeclining to levels indistinguishable from that from a promoterlessreporter construct (FIGS. 7 B&C). This suppression was reversed byreplenishing the GPBP-depleted nuclear extract with purified recombinantmGPBP (FIGS. 7 B&C). More recently, this finding of GPBP dependence fortranscriptional activity has been extended to the only additionalG+C-rich promoter tested (Yeung et al., unpublished data). This promotershared no sequence homology with the murine ADA gene's MSPE, but showeda similar secondary DNA structure forming potential (Ackerman et al.,1993), and can also bind purified recombinant mGPBP in EMSA experimentssimilar to those shown for the ADA gene's G+C-rich promoter (FIG. 4B).Additionally, six other additional mammalian G+C-rich promoters testedall showed a similar capacity to bind purified recombinant GPBP (Yeunget al., unpublished data). All these other G+C-rich promoters examinedalso displayed similar degrees of DNA sequence diversity and DNAsecondary structure forming potential similarity comparable to themurine ADA gene promoter's MSPE (Ackerman et al., 1993, and Yeung etal., unpublished data). These observations thus extend our mouse ADAgene promoter-based model and strongly support a proposed role for GPBPas a general transcription factor that can bind multiple mammalianG+C-rich promoters and mediate the assembly of transcription initiationcomplexes at these promoters.

In striking contrast to the G+C-rich promoter's requirement for GPBP inthe in vitro transcription assay, transcription activity from theadenovirus major late gene's classical TATAAA-box dependent promoter wastotally unaffected by the immunoabsorption mediated sequestering of GPBPin the HeLa cell nuclear extract (FIGS. 7 B&C). These resultsestablished that GPBP is only required for transcription initiated atthe G+C-rich promoter, but is totally dispensable for transcriptioninitiated at a classical TATAAA-box dependent promoter. The observedlinearity of GPBP's transactivation effect (FIG. 7A) and its absoluterequirement in G+C-rich promoter-directed transcription (FIG. 7C) areboth consistent with the predicted role of GPBP as a rate-limitingfactor for transcription initiated at a G+C-rich promoter.

Recently (Trembley, J. H. et al., 2002), the same in vitro transcriptionassays (with the same reporter constructs described above as templates)were used to examine transcription elongation suppression caused byimmunoabsorption of a factor that associates with members of each of thethree various classes (Conaway et al, 2000) of transcription elongationcomplexes. In contrast to the results described for the immunoabsorptionof GPBP, transcript production from both the G+C-rich andTATAAA-dependent promoters was similarly inhibited by the addition ofantibodies directed against that proposed transcription elongationfactor (Trembley, J. H. et al., in press). This observation suggeststhat elongation of transcripts initiated at both G+C-rich andTATAAA-dependent promoters may utilize common elongation complexes, andimplicate the differential transcription inhibitory effects displayed byGPBP-sequestering as interference against a differenttranscription-associated process. Since GPBP can bind to a G+C-richpromoter's MSPE, can associate with all the known transcriptioninitiation assembly factors tested, and is functionally required fortranscription initiated at that promoter, the anti-GPBP antibodies hadprobably interfered with a TATAAA-box independent transcriptioninitiation complex assembly step. Whether these anti-mGPBP antibodieswould interfere with transcription at non-G+C-rich, TATA-box deficientinitiator-element dependent promoters and whether GPBP is similarlyrequired for transcription initiated at all mammalian G+C-rich promotersremain unresolved questions that have now become tractable with the newreagents we now have on hand.

These results thus describe the discovery of a novel requisitepromoter-specific transactivating transcription factor with demonstrableabilities to both bind a typical G+C-rich promoter and interact withmultiple transcription factors that comprise the core mammalian RNApolymerase II transcription initiation complex. Since GPBP is notrequired for transcription directed by the adenovirus major late gene'sclassical TATAAA-box-dependent promoter, the results also providedefinitive proof that transcription initiated at the murine ADA gene'sG+C-rich promoter is mechanistically distinct from that initiated at aclassical TATAAA-box-dependent promoter.

1. Definitions

As used herein, the term “analog”, when used in the context of a peptideor polypeptide, means a peptide or polypeptide comprising one or morenon-standard amino acids or other structural variations from theconventional set of amino acids, and when used in the context of nucleicacids means a nucleic acid comprising one or more non-standardnucleotides or other structural variations from the conventional set ofnucleotides.

As used herein, the term “derivative”, when used in the context of apeptide, polypeptide, or nucleic acid means a peptide, polypeptide ornucleic acid different other than in primary structure (amino acids,amino acid analogs, nucleotides and nucleotide analogs). By way ofillustration, derivatives may differ by being glycosylated, one form ofpost-translational modification. For example, peptides or polypeptidesmay exhibit glycosylation patterns due to expression in heterologoussystems. If at least one biological activity is retained, then thesepeptides or polypeptides are derivatives according to the invention.Other derivatives include, but are not limited to, radiolabelledpeptides, polypeptides or nucleic acids, fusion peptides or fusionpolypeptides having a covalently modified N- or C-terminus, PEGylatedpeptides or polypeptides, peptides or polypeptides associated with lipidmoieties, alkylated peptides or polypeptides, peptides or polypeptideslinked via an amino acid side-chain functional group to other peptides,polypeptides or chemicals, and additional modifications as would beunderstood in the art.

As used herein, the term “fragment”, when used in the context of apeptide or polypeptide, means a peptide of from about 8 to about 50amino acids in length. The fragment may be 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50amino acids in length. When used in the context of a nucleic acid,“fragment” means a nucleic acid of from about 5 ti about 50 nucleotidesin length. The fragment may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50nucleotides in length.

As used herein, the term “homolog”, when used in the context of apeptide, polypeptide, or nucleic acid means a peptide, polypeptide ornucleic acid sharing a common evolutionary ancestor.

As used herein, the term “variant”, when used in the context of apeptide or polypeptide, means a peptide or polypeptide that differs inamino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. When used in the context of a nucleic acid, “variant” means anucleic acid that differs in nucleotide sequence by the insertion,deletion, or conservative substitution of nucleotides, but is able tohybridize to a sequence encoding GPBP, or complement thereof, understringent conditions. For purposes of the present invention, “biologicalactivity” includes, but is not limited to, the ability to be bound by aspecific antibody.

A conservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157: 105-132 (1982). The hydropathic index of an amino acid is based ona consideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of +2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, which isincorporated herein by reference. Substitution of amino acids havingsimilar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. In one aspect, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

Additionally, computerized algorithms are available to assist inpredicting amino acid sequence domains likely to be accessible to anaqueous solvent. These domains are known in the art to frequently bedisposed towards the exterior of a peptide, thereby potentiallycontributing to binding determinants, including antigenic determinants.

2. GPBP

The present invention provides GPBP-related materials and methods. GPBPpolypeptides may bind to a G+C-rich promoter. A representative exampleof a G+C-rich promoter includes, but is not limited to, MSPE

Representative examples of GPBP include, but are not limited to, murineGPBP and human GPBP. GPBP includes vasculin (SEQ ID NO:6), as disclosedby Bijnens et al. (2003). Vasculin is identical to human GPBP, exceptfor a 60 nucleotide exon that is missing in vasculin. The missing 60nucleotides in the cDNA encoding vasculin may be identified by aligningSEQ ID NO:3 and SEQ ID NO:5.

a. Nucleic Acids

The present invention is related to nucleic acids encoding GPBP, as wellas fragments, analogs, homologs and derivatives thereof. The nucleicacid may be DNA or RNA, single- or double-stranded, and may be maypurified and isolated from a native source, or produced using syntheticor recombinant techniques known in the art.

The present invention is also related to vectors comprising the nucleicacid. The vector may be an expression vector. The expression vectors maybe designed for expression of the polypeptide in prokaryotic oreukaryotic cells. The nucleic acid may be operative linked to a controlregion, which may regulate expression of the polypeptide. Representativeexamples of inducible expression systems are disclosed in Sambrook etal., Molecular Cloning, which is incorporated herein by reference.

The present invention is also related to host cells transformed ortransfected with the vector. Representative host cells are discussed inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990), the contents of which are herebyincorporated in their entirety.

b. Polypeptides

The present invention is also related to GPBP polypeptides, as well asfragments, analogs, homologs, variants and derivatives thereof. Thepolypeptides may be native or recombinant.

The present invention is also related to methods of making thepolypeptide. The polypeptide may be made by a method comprisingexpressing a nucleic acid encoding the polypeptide in a suitable hostcell and purifying the polypeptide. Methods for the optimization ofprotein expression in host cells are discussed further in Hannig et al.,Trends Biotechnol., 16(2):54-60 (1998), the contents of which are herebyincorporated by reference in their entirety. Other methods for makingthe polypeptide use techniques that are known in the art, such as theisolation and purification of native polypeptides or the use ofsynthetic techniques for polypeptide production.

c. Antibodies

The present invention is also related to antibodies that specificallybind to GPBP. The antibodies may be produced using techniques known inthe art. The antibody may be of classes IgG, IgM, IgA, IgD or IgE, orfragments or derivatives thereof, including Fab, F(ab′)₂, Fd, and singlechain antibodies, diabodies, bispecific antibodies, bifunctionalantibodies and derivatives thereof. The antibody may be a monoclonalantibody, polyclonal antibody, affinity purified antibody, or mixturesthereof which exhibits sufficient binding specificity to a desiredepitope or a sequence derived therefrom. The antibody may also be achimeric antibody, CDR-grafted antibody, “humanized” antibody, and otherantibody forms known in the art. The antibody may be derivatized by theattachment of one or more chemical, peptide, or polypeptide moietiesknown in the art. The antibody may be conjugated with a chemical moiety.

3. Diagnosis

The present invention is also related to methods of diagnosingconditions associated with altered expression of GPBP. Alteredexpression of GPBP in a sample may be diagnosed by contacting a samplewith an antibody that specifically binds to GPBP, measuring the binding,and comparing the binding to a control. Altered expression of GPBP maybe identified by a difference in the binding of the antibody to thesample and the control.

a. Cancer

A condition associated with altered expression of GPBP is cancer. GPBPmay be overexpressed in tumors since GPBP-regulated promoters areimportant in cell growth and differentiation. As discussed above,vasculin as reported by Bijnens et al. is identical to human GPBP,except for a 60 nucleotide exon that is missing in vasculin. An NCBIexpression profile search of vasculin shows that GPBP (vasculin) isoverexpressed in lung neuroendocrine tumors and inflamed endothelialcells in vascular and colon tissue. Differential expression analysisindicates that GPBP is also overexpressed in certain tumor cells (datanot shown). Furthermore, the Examples herein show that GPBP isoverexpressed in HeLa cells, which is a very metastatic tumor.

b. Atherogenesis

Another condition associated with altered expression of GPBP isatherogenesis. Bijnens et al. disclose that vasculin is involved inatherogenesis. As discussed above, vasculin as reported by Bijnens etal., is identical to human GPBP except for a 60 nucleotide exon that ismissing in vasculin.

4. Nanostructures

The present invention is also related to the use of GPMP polypeptidesfor stabilizing secondary DNA structures. DNA and DNA-protein complexesare emerging as powerful tools in nanotechnology. DNA nanostructureshave been constructed as nanomachines, nanoswitches, nanosensors,scaffolds and for barcodes for commercial and security purposes. The DNAnanostructures may be based on specific patterns of secondary structuresbased on base sequence. However, the stabilities of DNA secondarystructures are affected by local environment, including temperature,ionic environment, and the presence of neighboring sites with thepotential to form secondary structures. Regulating DNA secondarystructure may also be used in producing replicable nanomachines. Forexample, complex folded or branched DNA does not readily self-replicate,however, GPBP may be used to regulate secondary and tertiary structuresthat allow DNA to be cloned, for example, by using the polymerase chainreaction.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

EXAMPLE 1 Molecular Cloning of the Mouse G+C-Rich Promoter BindingProtein mGPBP

The murine ADA gene has an archetypal G+C-rich promoter that containsmultiple Sp1 binding sites and a non-canonical TATAAA-like elementTAAAAAA. Neither the Sp1 binding sites nor the TAAAAAA sequence arerequired for basal or enhancer-activated promoter function (Ackerman etal, 1993, Hsu et al., submitted). Instead, the minimal self-sufficientpromoter element (MSPE) within the murine ADA gene promoter hadpreviously been shown to reside within a 48 bp element “C” whichdisplays an imperfect dyad sequence symmetry (FIG. 1 of Ackerman et al.,1993). Moreover, Electrophoretic Mobility Shift Assays (EMSA) coupledwith DMS footprinting had revealed that a nuclear protein(s) could bindto this element (Ackerman et al., 1993). Many other G+C-rich promotersalso contain elements with similar secondary structure-forming potentialaround their transcription initiation sites (Ackerman et al., 1993). Wepreviously demonstrated that there are nuclear protein-binding siteswithin the ADA gene's MSPE (Ackerman et al., 1993). This observation ledus to attempt the cloning and characterization of the nuclear proteinresponsible for directing the assembly of the RNA polymerase IItranscription initiation complex at this promoter.

We used a ³²P-labeled multimerized ADA promoter fragment C (Ackerman etal., 1993) as a probe to screen a λgt11 bacteriophage mouse brain cDNAexpression library for cloned proteins that bind to this MSPE. Therestriction fragment C was isolated from plasmid, gel-purified andmultimerized by ligating end to end in the presence of T4 ligase.Multimers containing 4-5 copies of the fragment were gel-purified,end-labeled with polynucleotide kinase and γ³²P-ATP and used to screenthe expression library according to the method of Singh et al., 1988.Screening with the negative control Sp1 binding consensus motif probe(TGGGCGGGGC) (SEQ ID NO:11) eliminated clones expressing proteins thatbind nonspecifically to random G+C rich sequences. Out of 3×10⁷ clones,2 clones were identified after tertiary screens and these were furtherexamined by subcloning, restriction digestion and sequence analysis.

Sequence analysis of the subcloned cDNA fragments derived from the twoclones revealed that one cDNA insert fragment contained an open readingframe (ORF) which shared regions of sequence homology with several knowntranscription factors. These factors include yeast TFIIFα, whichinteracts with RNA polymerase II in transcription initiation complexassembly (Aso et al., 1992, Killeen & Greenblatt, 1992); yeast ARSbinding factor 1 (yABF-1), which is a known transcription factorinvolved in DNA replication initiation in yeast (Biswas et al., 1990,Martens & Brandl, 1994); mouse high mobility group-1-like StructureSpecific Recognition Protein 1 (SSRP1), which apparently binds to DNAthrough structural recognition (Shirakata et al., 1991, Bruhn et al.1992) and participates in both transcription activation (Dyer et al.,1998, Spencer et al., 1999) and elongation (Orphanides et al., 1999);and helicases, which can unwind double-stranded DNA for transcriptionand replication (recently reviewed by van Brabant et al., 2000).

Based on our expectations of the properties of the gene sought, thisfragment was deemed to be a good candidate clone and was used togenerate 2 full-length cDNA clones, 3.0 and 3.5 kb in size, by 3′ and 5′rapid amplification of cDNA ends (RACE). RACE was performed usingmarathon-ready mouse brain cDNA templates and Advantage® cDNA PCR kitmanufacturer (Clontech) according to recommended protocols. For the 5′RACE, the primary PCR reaction used primer5′-CAGGCTGGAGCAAAGTCATGCTGCGCC-3′ (SEQ ID NO:7) with the AP1 primer andthe nested PCR reaction used primer 5′-AGGTCCAGTCTCTCCAACTCAGTGAAAC-3′(SEQ ID NO:8) and the AP2 primer. For the 3′ RACE, the primary PCRreaction used primer 5′-CTGATCTGTGACTTCAAGTTTGGACC-3′ (SEQ ID NO:9) withthe AP1 primer and the nested PCR reaction used primer5′-TGACGATGTGTGAAGGAGATCCTCACAGC-3′ (SEQ ID NO:10) with the AP2 primer.The entire cDNA sequence of the 3,451-bp clone, including the deduced493 amino acid sequence encoded by the 1,479 bp ORF, and the 815 bp3′UTR, is shown in FIG. 1A. This cloned gene was named the murineG+C-rich Promoter Binding Protein (mGPBP) gene.

EXAMPLE 2 The mGPBP Transcript is Ubiquitously Expressed as 2 mGPBPSpecies Differing in 3′-Polyadenylation Site Utilization

Because G+C-rich promoters control practically all housekeeping genes,which are essential for cell survival, we predicted that if mGPBP iscritical for transcription of these genes, it would be ubiquitouslyexpressed. The full-length mGPBP cDNA sequence was labeled and used as aprobe to determine the tissue distribution and mRNA sizes of the gene ina Northern blot analysis.

Mouse Multiple Tissue Northern (MTN™) Blots (Clontech) were hybridizedwith murine GPBP cDNA probes and washed under standard conditions(Sambrook et al., 1989). The probes used were gel-purified, individuallycloned mGPBP cDNA restriction fragments. The blots used were stripped aspreviously described (Gum, R. et al., submitted) and reprobed with aβ-actin cDNA probe as a loading control. All probes were synthesized bythe random hexanucleotide-priming method (Feinberg and Vogelstein, 1984)and purified using a Bio-Gel P-30 column (BIO-RAD).

The results (FIG. 2A) showed that the gene is highly expressed in allmouse tissues analyzed as 2 mRNA species sized at 3.0 and 3.5 kb,respectively. Because we had two 3′ RACE products that differed in sizeby ˜500 bp, and each product contained consensus AATAAA polyadenylationsignaling motifs located appropriately upstream of the poly-A tracts, wesurmised that the 3.0 and 3.5 kb mRNA species may represent the same RNAwith different polyadenylation site utilization. This prediction wasconfirmed when we cloned out a 2.0 kb fragment from the 5′ end and a 350bp fragment from the 3′ end of the longer cDNA clone and used them asprobes, respectively, on the same Northern blot which was strippedbetween each hybridization experiment. The results (FIG. 2B) revealedthat the 5′ probe hybridized with both the 3.0 and 3.5 kb mRNA species,whereas the 3′ probe hybridized only with the 3.5 kb mRNA species. Theseobservations indicate that both these cross-hybridizing mRNAs containthe entire ORF of the mGPBP gene and differ only in their choice of 3′polyadenylation site usage.

EXAMPLE 3 Expression of Recombinant mGPBP

The murine GPBP cDNA's ORF sequence was inserted downstream of thehexa-histidine tag sequence of pET28a (Novagen) to yield the bacterialexpression vector pETmGPBP that can synthesize recombinant his-taggedmGPBP upon IPTG induction. The bacterial expression vector used tosynthesize recombinant GST-mGPBP fusion protein was generated byinserting the mGPBP cDNA's ORF downstream of the glutathioneS-transferase (GST) cDNA sequence in plasmid pGEX-4T-1 (Pharmacia) toyield construct pGEX-mGPBP. These bacterial expression constructs weretransformed into E. coli strains BL21/DE3 and BL21 for the production ofrecombinant mGPBP, respectively. The eukaryotic mGPBP expression vectorpCDNA-mGPBP was generated by inserting the mGPBP cDNA's ORF into thepCDNA3 expression vector (Invitrogen). The hemagglutinin antigen(HA)-tagged (Field, 1988[2]) mGPBP eukaryotic expression constructpCDNAHA-mGPBP was generated by inserting the (HA) tag coding sequenceupstream of, and in frame with, the mGPBP cDNA's ORF in pCDNA-mGPBP.

The 1,479 bp ORF of the mGPBP cDNA clone was inserted downstream of theIPTG-inducible promoter as an in-frame histidine-tagged fusion proteinencoding sequence in the PET28a bacterial expression plasmid. Theresultant pETmGPBP plasmid was introduced into the E. coli BL21/DE3strain host for recombinant mGPBP synthesis. Cell lysates fromIPTG-induced and IPTG-uninduced transformed BL21 cells were prepared andanalyzed by SDS polyacrylamide gel electrophoresis (PAGE) and CoomassieBlue staining. Cell lysates from IPTG-induced cells displayed aprominent 66 kD protein band that was absent from IPTG-uninduced cells(FIG. 3A, lanes 3&2, respectively). The 1,479 bp ORF of the mGPBP cDNAclone was also inserted into pCDNA3 (Invitrogen), a eukaryoticexpression vector. Expression of the ORF in reticulocyte lysates alsoresulted in the synthesis of a novel 66 kD protein.

Recombinant His-tagged mGPBP was purified under native conditions usingQiagen's Ni-NTA agarose following the manufacturer's protocol with minormodifications. After incubation of bacterial lysate with a 50% Ni-NTAagarose slurry at 4° C. for 2 hours, the resin-bound protein was elutedfrom the agarose matrix and the protein fractions containing thepurified protein were then pooled and dialyzed twice for 60 minuteseach, first against 500 ml of dialysis buffer (20 mM Tris-HCl, pH7.5, 50mM KCl, 10 mM MgCl₂, 10 μM ZnSO₄, 1 mM EDTA, 20% glycerol, 0.5 mM DTT,0.2 mM PMSF) containing 0.25M NaCl, and then against 500 ml of the samedialysis buffer without NaCl. The his-mGPBP fusion protein purified byNi resin affinity column chromatography and SDS-PAGE was found tobe >95% pure as revealed by SDS-PAGE and Coomassie Blue staininganalysis (FIG. 3A, lane 4).

EXAMPLE 4 Inhibitory Effect of UTR

The presence of all or part of the 1.15 kb 5′ UTR in the mGPBP mRNA hadan inhibitory effect in the translation of this mRNA in both expressionsystems: bacteria (FIG. 3) and reticulocyte lysates (data not shown).However, only a single 66 kd translation product was producedirrespective of the size of the 5′ UTR present in the mRNA (data notshown). The 3′ UTR of the GPBP mRNA contains multiple TAAAT repeats(FIG. 1A) that are reportedly involved in mRNA turnover control (Shawand Kamen, 1986). The presence of either the long or short 3′UTR alsohad no effect on the size of the translation product generated from themRNA using either prokaryotic or mammalian in vitro translation systems(data not shown).

EXAMPLE 5 Production of Antibodies to mGPBP

Recombinant mGPBP (His-tagged) used to raise anti-mGPBP antiserum waspurified under denaturing conditions using Ni-NTA agarose (Qiagen)following the vendor's instruction and then isolated as a single excisedband following electrophoresis in 8.5% sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE). The recovered mGPBP protein wasconcentrated by using ULTRAFREE®-MC 10,000NMWL filter units (Millipore).

Purified his-mGPBP was used to raise rabbit polyclonal antiserum againstmGPBP. Immunization was performed at the Immunological Resource Centerat the University of Illinois. Antibodies from the antiserum werepurified by using the ImmunoPure® (A/G) IgG purification kit (Pierce),as suggested by the manufacturer. Eluted antibodies were combined,dialyzed against PBS and concentrated with centrifugal filtration units(Millipore).

The mGPBP antiserum was shown by Western blot analyses to beimmunoreactive against mGPBP in either IPTG-induced pETmGPBP carryingBL21 cell lysate or purified mGPBP preparations (FIG. 3A, lanes 7&8,respectively) and showed no cross-reactivity with BL21 bacterialproteins (FIG. 3A, lane 6).

EXAMPLE 6 Expression Pattern of mGPBP

The anti-mGPBP antiserum was used to examine the presence of GPBP inseveral human and mouse cell lines. Cells were lysed in RIPA buffer (25mM Tris pH 8.2, 50 mM NaCl, 0.5% Nonidet P40, 0.5% sodium deoxcholate,0.1% SDS, 0.1% sodium azide) containing 1 mM PMSF, 10 μg/ml of aprotininand 10 μg/ml of leupeptin. Cell lysates were incubated on ice for 15minutes and centrifuged at 12,000×g for 10 min. at 4° C. The proteinconcentration of the cleared lysates was determined using the BioRadprotein assay kit. Equal amounts of protein per lane were analyzed in8.5% SDS-PAGE. Western blotting was carried out using Amersham PVDFmembranes according to the vendor's recommendations.

All of the cell lysates analyzed by Western blotting using theanti-mGPBP antiserum as probe revealed a prominent 66 kD band (FIG. 3B).Since the cells used were of both mouse (FIG. 3B, lanes 2&5) and human(FIG. 3B, lanes 1, 3, 4&6) origin, the results indicate that ouranti-mGPBP antiserum is immunoreactive with both mouse and human GPBP.This conclusion was confirmed by subsequent Western blot analysesperformed on purified and recombinant human GPBP, which is encoded by asimilarly sized ORF in our full-length human GPBP cDNA clone (FIG. 8).Thus the ORF within our full-length mGPBP cDNA clone does encode a 66 kDprotein and the translation product of that ORF in either bacterial ormammalian cells was immunoreactive to our anti-mGPBP antiserum. Tissuedistribution analysis confirmed that GPBP is present in all mammaliantissues and cells examined to date, as predicted for a candidatecritical regulator of housekeeping gene transcription.

EXAMPLE 7 Recombinant mGPBP can Bind Specifically to the MSPE of theMouse ADA Gene

The DNA binding capability of mGPBP was examined directly in EMSAs usingeither the multimerized fragment C′ with imperfect dyad symmetry (seeFIG. 5 of Ackerman et al., 1993) or the 236 bp murine ADA gene promoteras probe. The probes were isolated as restriction fragments fromplasmids and radiolabeled with α³²P-dCTP using Klenow fragment.Electrophoretic mobility shift assays were performed as previouslydescribed (Christy and Nathans, 1989) with minor modifications.Binding-reaction mixtures containing purified bacterially expressedrecombinant proteins were prepared in binding buffer (10 mM Tris-HClpH7.5, 60 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA, 10 μM ZnCl₂,0.05% NP-40, 12-15% glycerol) containing 0.5 μg of poly-dI/dC (20 μltotal volume). After the binding reaction mixtures had been incubatedfor 10 minutes at room temperature, the radiolabeled probe was added foran additional 20 minutes of incubation at room temperature. The mixturewas then separated in 4% (acrylamide/bis-acrylamide-29:1) non-denaturingpolyacrylamide gel containing 1×TBE buffer.

The assay results are shown in FIG. 4. In panel A, the labeled DNA probeused consisted of 4 tandem-repeat copies of fragment C′ (4C′) that hadbeen end-ligated. Purified recombinant mGPBP bound specifically to thisprobe and retarded probe electrophoretic mobility (FIG. 4A, lanes 1 &2).This binding was specifically competed out by adding excess unlabeledprobes (lanes 3 & 4), but not by adding similar amounts of unlabeled E2Fbinding motif (lanes 5 & 6) or a 200 bp plasmid sequence (lanes 7 & 8).As indicated in panel B, a single copy of this fragment C′ in thecontext of the labeled 236 bp mouse ADA gene promoter also bound topurified recombinant mGPBP (lanes 1 & 2). This binding was competed outby excess unlabeled probe (lanes 3 & 4) or by excess amounts of theunlabeled tandem repeated C′ fragment (4C′) probe used in FIG. 4A (lanes5 & 6). Again, this binding was not competed out by similar excessamounts of unlabeled E2F binding sequence (lanes 7 & 8) or the 200 bpplasmid sequences (lanes 9 & 10). In similar gel mobility shift assaysusing the 200 bp plasmid sequences as probe, mGPBP was unable to bind toand retard the mobility of this nonspecific DNA probe (data not shown).These results demonstrate that recombinant mGPBP can bind specificallyto the 48 bp MSPE located within the 236 bp murine ADA gene promoter.

EXAMPLE 8 GPBP is a Nuclear Protein

Since cellular transcription factors all function within the cellnucleus, we used indirect immunofluorescence to examine the cellularlocalization of the GPBP. This was accomplished by using protein-Aaffinity-purified antibodies derived from our anti-mGPBP antiserum αN-80to immunostain fixed and permeablized human HeLa cells, which were thencounter-stained with DAPI to locate the nuclei.

5×10⁵ Hela cells were plated in DMEM containing 10% fetal calf serum in60 mm dishes containing glass coverslips and incubated at 37° C.overnight. The following day the cells were transfected with 5 μg ofeither the pCDNAHA-mGPBP expression construct or the pCDNA-HA controlvector using a calcium phosphate transfection protocol as describedpreviously (Ackerman et al., 1993). The hemagglutinin antigen(HA)-tagged (Field, 1988[2]) mGPBP eukaryotic expression constructpCDNAHA-mGPBP was generated by inserting the (HA) tag coding sequenceupstream of, and in frame with, the mGPBP cDNA's ORF in pCDNA-mGPBP.Sixteen hours after transfection, the cells were rinsed extensively withPBS, treated with fresh medium and incubated for an additional 48 hours.The cells were then rinsed with PBS and fixed with 4% paraformaldehydefor 30 minutes at room temperature. After fixation the cells werepermeablized with 0.2% Triton X-100/PBS for 5 minutes, rinsed andstained with either mGPBP antibodies, or preimmune antisera derivedantibodies that had been purified by binding to protein A-conjugatedbeads, or antibody against HA (2 μg/ml) at 37° C. for 1 hour. Afterwashing 4 times (5 minutes/wash) with PBS the cells were stained witheither rabbit or mouse fluorescein-conjugated secondary antibodies(1:200 dilution) (Amersham) at 37° C. for an additional hour. Thecoverslips were then washed extensively, mounted in antifade solution(Vector Lab) containing 0.25 μg/ml DAPI to counterstain the nuclei andphotographed using a Zeiss Axiovert microscope with an attachedPrinceton Instruments CCD camera.

The anti-mGPBP signal was nuclear-localized (FIG. 5A). In controlexperiments using protein-A affinity-purified antibodies derived frompre-immune antiserum as a probe, immuno-reactive signals were negligibleeven upon prolonged photographic exposure (FIG. 5B). The nuclearlocalization of GPBP was confirmed by directly transfecting the mGPBPexpression vector into human HeLa cells and immunostaining for mGPBPusing our anti-mGPBP antibodies (FIG. 5C, top panel). Definitive proofthat our recombinant mGPBP was nuclear-localized was provided bytransfecting an mGPBP-HA-tag fusion protein expression vector into humanHeLa cells and immunostaining for the tagged protein with anti-HAantibodies (FIG. 5C bottom panel). These in situ immunohistochemicalanalyses demonstrated that both the endogenous human GPBP and theexogenous cDNA-encoded recombinant mGPBP could translocate into the cellnucleus.

EXAMPLE 9 In Vitro complex formation of GPBP with Multiple Key Factorsthat Participate in Mammalian RNA-Polymerase II Transcription InitiationComplex Assembly

If GPBP binding to the G+C-rich promoter's MSPE can lead to the assemblyof the transcription initiation complex at that location, GPBP should beable to interact with one or more transcription factors that normallyparticipate in transcription initiation complex formation. Thisexpectation was tested by assaying whether immobilized recombinantGST-mGPBP fusion protein could complex with various nucleartranscription factors of the RNA polymerase II transcription initiationcomplex, present in nuclear extracts.

The bacterial expression vector used to synthesize recombinant GST-mGPBPfusion protein was generated by inserting the mGPBP cDNA's ORFdownstream of the glutathione S-transferase (GST) cDNA sequence inplasmid pGEX-4T-1 (Pharmacia) to yield construct pGEX-mGPBP. Thisbacterial expression constructs were transformed into E. coli strainsBL21 for the production of recombinant mGPBP. The GST-mGPBP fusionprotein was purified according to a published protocol (Guan and Dixon,1991) with minor modifications. After IPTG induction, the bacterialcells were pelleted and resuspended in PBS containing 10 mMDTT, 0.5 mMPMSF and 2 μg/ml each of leupeptin, pepstain A, and antipain, sonicated,and subsequently treated with 1/10 volume of 10% Triton X-100. TheGST-fusion protein in the supernatant was then purified by one stepaffinity chromatography using glutathione-sepharose beads (Pharmacia)according to the vendor's recommendations.

Nuclear extracts were prepared as previously described (Berger andKimmel, 1987) with minor modifications. Mouse C1-1D Cells were collectedand washed with 10 volumes of PBS once. The cells were then suspended in5 volumes of cold buffer A (10 mM Hepes-KOH pH 7.9, mM KCL, 1.5 mMMgCl₂, 0.1 mM EGTA, 0.5 mM DTT, 0.5 mM PMSF, 2 μg/ml each of antipain,leupeptin, and pepstain) and incubated on ice for 15 minutes. Cells werelysed using 20 strokes of a Dounce homogenizer (B pestle), and a further2-5 strokes in the presence of 0.3-0.4% Nonidet-P40 (NP-40). The lysedcells were centrifuged at 1,300×g for 10 minutes. The pelleted nucleiwere washed thrice with buffer A without NP-40, centrifuged again asabove, and resuspended in 1-2 volumes of cold buffer B (10 mM HEPES-KOHpH7.9, 0.1 mM EGTA, 0.5 mM DTT, 400 mM NaCl, 5% glycerol, 0.5 mM PMSF)and incubated on ice for 60 minutes. The solution was then centrifugedat 48,000×g for 1 hour. The supernatant was dialyzed twice against 500ml of dialysis buffer (20 mM HEPES-KOH pH7.9, 75 mM NaCl, 0.1 mM EDTA,0.5 mM DTT, 20% glycerol, 0.5 mM PMSF) for 1 hr., cleared bycentrifugation in a microcentrifuge for 15 min.

Recombinant mGPBP fused in-frame to the GST tag was immobilized bybinding the GST moiety to glutathione covalently linked to beads. 1-5 μgof the fusion protein was incubated with 100 μl of a 50% slurry ofglutathione-Sepharose beads (Pharmacia) for 1 hour at 4° C. Theprotein-bound beads were collected and washed three times with PBSbuffer. The beads were then re-suspended in binding buffer (20 mMHepes-KOH pH7.9, 75 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 20% glycerol, 0.5mM PMSF) (Hayes et al., 1998). About 1/60 of the beads were subjected to8.5% SDS-PAGE to confirm that GST proteins were bound to the beads.Mouse C1-1D nuclear extract proteins were then allowed to bind to thebead-immobilized mGPBP by incubating the rest of the beads with 100 μgof C1-1D cell nuclear extract on a rotator overnight at 4° C. The beadswere collected and washed three times with washing buffer (20 mMTris-HCl, pH8.0, 150 mM NaCl, 1 mM EDTA, and 0.1-0.3% NP-40). The boundproteins were eluted with SDS-sample buffer and subjected to 4-20%SDS-PAGE. Western blotting was then carried out on the gels. Both theunbound proteins in the supernatant and the proteins that remained boundto the immobilized mGPBP after extensive washing were then analyzed byWestern blotting using antibodies against various transcription factorsas probes.

The results showed that known transcription initiation complex factorssuch as the TATA-binding Protein (TBP), Transcription Fraction IIB(TFIIB), Transcription Fraction IIF (TFIIF RAP30), and RNA polymerase II(RNA pol II CTD), as well as the transcription factor P300/CAAT BindingProtein (CBP), all complexed with the immobilized recombinant mGPBP(FIG. 6A). None of these same proteins complexed with only the GST tagimmobilized onto the same glutathione beads, and the unbound supernatantrecovered from both binding assays showed that the same proteins weresimilarly present in both binding assays. The nuclear membrane proteinnucleoporin p62, which does not participate in transcription initiationcomplex formation, showed no affinity to either immobilized mGPGP or GST(FIG. 6A). These in vitro results demonstrate that mGPBP can complexspecifically with several key transcription initiation factors.

EXAMPLE 10 In Vivo Complex Formation of GPBP with Multiple Key Factorsthat Participate in Mammalian RNA-Polymerase II Transcription InitiationComplex Assembly

To examine whether the in vitro complexing of GPBP with transcriptionfactors also occurs in vivo, we performed a co-immunoprecipitationexperiment using an HA-tagged mGPBP expression construct in mouse C1-1Dcells. The hemagglutinin antigen (HA)-tagged (Field, 1988[2]) mGPBPeukaryotic expression construct pCDNAHA-mGPBP was generated by insertingthe (HA) tag coding sequence upstream of, and in frame with, the mGPBPcDNA's ORF in pCDNA-mGPBP. Anti-HA antibodies were used toimmunoprecipitate nuclear extract proteins derived from the transfectedcells. As a control, nuclear extract from cells transfected with the HAexpression vector containing no GPBP-encoding sequences underwent asimilar immunoprecipitation procedure.

Cells were collected by centrifugation in PBS 48 hours after DNAtransfection. Cells from each 100-mm dish were treated with 60 μl oflysis buffer containing 20 mM HEPES pH7.9, 400 mM NaCl, 1 mM EDTA, 0.1%NP-40, 1 mM DTT, 0.5 mM PMSF, 10% glycerol and 1.5 μl of proteaseinhibitors cocktail (Sigma) (Hayes et al., 1998). Cell lysates wereincubated for 30 minutes at 4° C., and centrifuged at 13,000×g for 10min. at 4° C. The supernatant from each 100-mm dish was treated with 300μl of lysis buffer without NaCl, and incubated with agarose bead-boundanti-HA antibody (Santa Cruz) overnight at 4° C. The beads were pelletedand washed 3-4 times with 1 ml of buffer W (20 mM Tris-HCl, pH8.0, 100mM NaCl, 0.1-0.3% Np-40, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF).

The proteins that co-immunoprecipitated with anti-HA antibodies werethen analyzed by Western blotting with a 4%-20% precast SDS-PAGE(BioWittaker Molecular Applications) using antibodies against TBP,TFIIB, CBP, and RNA polymerase II as probes. The antibodies used wereanti-HA monoclonal antibodies (clone 12CA5, Boehringer Mannheim);anti-RNAPII CTD (RNA polymerase II C-terminal Domain) monoclonalantibodies (Promega); anti-HA monoclonal antibodies conjugated withagarose, rabbit anti-TFIIB polyclonal antibodies C-18, rabbit anti-TFIIFRAP30 polyclonal antibodies C-17, anti-TBP monoclonal antibodies 58C9,rabbit anti-CBP polyclonal antibodies A-22 and WT rabbit polyclonalantibodies 180 (Santa Cruz); and anti-Nucleoporin p62 monoclonalantibodies clone 53 (Transduction Laboratories).

A portion of the respective nuclear extracts prior to treatment withanti-HA antibodies (input lysate, FIG. 6B) were shown by Westernblotting to contain all the requisite proteins. None of the nucleartranscription factors of interest co-immunoprecipitated with the HA tagalone. In contrast, TBP, TFIIB, CBP, RNA polymerase II (FIG. 6B) andTFIIF RAP30 (data not shown) all co-immunoprecipitated with theHA-tagged mGPBP. These results demonstrate that GPBP does complex withthese transcription factors both in vitro and in vivo.

EXAMPLE 11 Transcription from the Mouse ADA Gene's G+C-Rich PromoterRequires GPBP

To examine whether our cloned mGPBP could trans-activate a luciferasereporter gene controlled by the mouse ADA gene's G+C-rich promoter, weco-transfected both mouse C1-1D cells and human 293 cells with aconstant amount of the reporter construct and increasing amounts of themGPBP expression vector.

Monolayer cultures of murine C1-1D LM (TK⁻) fibroblast cells derivedfrom bone marrow stromal cells of a (C57B1/6J×C3H/HeJ) F1 mouse, andhuman embryonic kidney (HEK) 293 cells were maintained in 10% fetal calfserum in DMEM (Dulbecco's Modified Eagle's Medium). For each 60-mm plateof C1-1D or 293 cells, 0.2 μg of the luciferase reporter gene under thecontrol of the murine ADA gene promoter was co-transfected with variousamounts of murine GPBP-expression plasmid (pCDNA-mGPBP) usingLipofectamine Plus (for 3 hours), as suggested by the manufacturer(Gibco BRL). The total amount of plasmid used per plate was brought upto 2 μg with the empty pCDNA3 vector. The transfected cells were washedwith PBS and then cultured in 10% FCS in DMEM medium for 24-36 hoursprior to being harvested for reporter gene expression analyses. Alltransfection assays were repeated at least 3 times using different DNApreparations. The luciferase activities of the transfected cell lysateswere measured using a luciferase assay system (Promega) and the VICTO²™Multilabel Counter (Wallac). The obtained values were then normalizedaccording to the protein concentrations. (Bio-Rad Protein Assay). Forboth C1-1D and 293 cell lines, reporter gene expression increasedlinearly with the amount of the mGPBP expression vector added in a dosedependent manner (FIG. 7A).

To address the question of whether GPBP is specifically required fortranscription directed by the murine ADA gene's G+C-rich promoter, weperformed in vitro transcription assays. In-vitro transcriptionreactions were carried out as previously described (Dignam et al., 1983)with minor modifications. Supercoiled template DNA was purified bybanding twice in centrifuged CsCl gradients (Sambrook et al., 1989). Thetemplates used consist of G-less cassette reporters which were eitherwithout a promoter (construct pC₂AT-Sawadogo and Roeder, 1985), with theadenovirus major late promoter (construct PMLC₂AT, Sawadogo and Roeder,1985) or with the murine ADA gene promoter (pmADAPC₂AT-generated byPCR). Transcription reaction mixtures (25 μl total volume) containing 8mM HEPES (pH 7.9), 40 mM KCl, 6 mM MgCl₂, 0.08 mM EDTA, 0.2 mM DTT, 8%glycerol, 30 units of RNase Ti, 100 ng template DNA, 9-10 μg HeLanuclear extract (Promega), and different amounts of antibodies andpurified recombinant protein were incubated at 30° C. for 10 minutesprior to the addition of 0.2 mM of ATP, CTP, and GTP to 0.2 mM each, UTPto 8 nM, 3′-O-Methyl-guanosine 5′-triphosphate (Pharmacia) to 0.05 mM,and α-³²P-UTP (800 Ci/mmole) to 1 μM. The reaction mixtures were thenincubated for 1 hour at 30° C. The reactions were stopped by theaddition of 175 μl of stop solution (0.3 M Tris-HCl, pH7.4, 0.3 M sodiumacetate, 0.5% SDS, 2 mM EDTA, 25 μg/ml tRNA) and an extraction control200 bp end-labeled DNA fragment. The reaction solutions were extractedtwice with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v). Thenucleic acids in the reaction were ethanol precipitated, air-dried anddissolved in 4 μl of Nuclease-Free water. An equal volume of loading dye(98% formamide, 10 mM EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue)was added to the in vitro transcribed RNA solution. These RNA solutionswere then heated at 90° C. for 10 minutes and electrophoresed in a 6%denaturing (7 M urea) polyacrylamide gel containing 0.5×TBE buffer. Theamounts of radiolabeled transcript and extraction control bands werethen quantified using a Phosphorimager (Molecular Dynamic).

The transcription assays were performed in the presence of HeLa cellnuclear extracts from which GPBP had been incrementally sequestered bythe addition of increasing amounts of anti-mGPBP antibodies that cancross-react with human GPBP. Recovery of the reporter transcripts fromthe transcription reaction mix was monitored using a labeled carriercontrol DNA. FIG. 7B shows a representative result from experiments thatwere repeated three or more times. In the presence of increasing amountsof anti-mGPBP antibodies, there was a corresponding decrease intranscript production (FIG. 7B, lanes 4-9). When increasing amounts ofpurified recombinant mGPBP were added back to the antibody-treatednuclear extract, the observed antibody-induced suppression of reportergene transcription was correspondingly reversed (FIG. 6B, lanes 10 and11). Control experiments revealed that pre-immune serum antibodies hadno suppressive effect on reporter gene transcription (FIG. 7B, lanes 3,2, &1). The suppressive effect of anti-mGPBP antibodies on transcriptionwas observed only when the reporter gene was under the control of theG+C-rich promoter. Transcription of the same reporter gene under thecontrol of the adenovirus-major late gene's TATAAA-box dependentpromoter was not diminished by the immuno-sequestering of GPBP in thenuclear extract (FIG. 7B, lanes 12-15). Quantifying all the labeledbands in several experiments using a phosphorimager allowed us todemonstrate the reproducibility of the results as summarized in FIG. 7C.These results indicate that although GPBP is required for transcriptiondirected by the ADA gene's G+C-rich promoter, it is not required fortranscription directed by the adenovirus Major late gene's classicalTATAAA-box dependent promoter.

EXAMPLE 12 The G+C-Rich TopoIIa Gene Promoter Binds Specifically tomGPBP

To examine whether mGPBP is a general requisite G+C-richpromoter-dependent transcription factor, we also examined whether thisprotein can bind to other G+C-rich promoters, especially one thatcontains a consensus TATA box and G+C-rich rich regions that display noobvious homology to the murine ADA gene promoter, as in the case of thehuman topoisomerase IIα promoter (FIG. 9A). EMSAs using the labeledTopoIIa promoter as the probe showed that the TopoIIa gene promoter canalso bind specifically to purified recombinant mGPBP in the absence ofany other mammalian transcription factors, This binding can be competedout by either excess unlabeled probe or excess unlabelled fragmentscontaining four copies of the murine Ada gene MSPE but not by excesscopies of a 100-bp fragment derived from the pUC2H vector, which doesnot adopt non-B form DNA structures under negative supercoilingconditions.

EXAMPLE 13 Transcription Initiation can be Only Partially Suppressed byImmunosequestration of mGPBP

In HeLa cell nuclear extract-dependent in vitro transcription assays, wedemonstrated that transcription initiation at the murine Ada genepromoter required the presence of GPBP whereas transcription initiationat a consensus TATA box-dependent adenovirus major late gene promoterdid not. Since this TopoIIa gene promoter binds specifically to mGPBPbut contains a canonical TATA box, we also examined how this promoterfunctioned when the GPBP in the nuclear extract was sequestered byimmunoabsorption. Sequestering of GPBP in the HeLa nuclear extract withanti-GPBP antibodies under conditions that totally suppressestranscription initiated by the murine Ada gene promoter showed only apartial suppressive effect on transcription initiation by the TopoIIagene promoter (FIG. 9C). This suppression can also be fully reverse bythe addition of purified recombinant mGPBP to the nuclear extract. Theresults in FIG. 9 thus demonstrated that mGPBP can indeed bind to otherG+C-rich promoters and that the presence of a canonical TATA boxrendered the TopoIIa gene promoter only partially dependent on GPBP fortranscription initiation, in contrast to the total dependence of themurine Ada gene promoter on GPBP for transcription in the absence ofsuch a canonical element.

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1. An isolated DNA encoding GPBP polypeptide, said polypeptidecomprising a sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4 and homologs thereof.
 2. The DNA of claim 1, wherein saidDNA comprises the sequence set forth in SEQ ID NO:3.
 3. A vector whichcomprises the DNA of claim 1 or claim 2, wherein said DNA is operativelylinked to a control region.
 4. The vector of claim 3, wherein saidvector is an expression vector.
 5. A host cell comprising the vector ofclaim
 3. 6. A host cell comprising the vector of claim 4.